Over the past two decades, the advent of high throughput experimentation has transformed the way life science and biomedical research is carried out. A convergence of technologies in fields such as genetic engineering, organic chemistry, materials science, microfabrication and microelectronics has led to new technology platforms (e.g. microarray technologies, microfluidic devices and systems, bead-based combinatorial compound libraries and assay systems, and microplate-based assay systems) to address applications ranging from high throughput screening of compound libraries for drug discovery to rapid whole genome sequencing.
Examples of high throughput screening systems for drug discovery include microfluidics-based platform technologies for running continuous-flow assays (e.g. receptor-ligand binding assays and cell-based assays for identifying receptor agonists), and microplate-based systems in which binding reactions, enzymatic reactions, or cell-based assays are run in a microwell plate format, and automated liquid-dispensing stations and plate-handling robotics provide for automated sample preparation, assay, and detection steps. The majority of existing high throughput platform technologies for drug discovery utilize fluorescence-based optical detection. Although fluorescence techniques provide for very high detection sensitivity, and are generally much more environmentally friendly than the more traditional, radioisotope-based approaches that predominated in biological assay methodologies of two decades ago, there are a number of drawbacks to the use of fluorescence. Examples include: (i) the requirement for sophisticated light sources, detectors, and optical systems, the performance of which are often sensitive to misalignment or instrumental drift, and (ii) photo-bleaching phenomena, which may result in degradation of signal over time in samples subjected to repeat measurements.
Another, more serious limitation of existing high throughput screening technologies stems from the growing awareness that a number of potential therapeutic targets, e.g. potential cancer therapeutic targets, that are attractive targets from a biological perspective are intractable (“undruggable”) from a chemical standpoint because they are generally not amenable to conventional drug discovery approaches. These protein targets typically possess a relatively large contact area when interacting with other proteins (i.e. through protein-protein interactions) or due to the fact that they possess a ligand that binds with extremely high affinity to the active site of the protein. In either case, finding a conventional small molecule or biologic (protein) drug candidate that will block the interaction (i.e. interfere with and/or obscure the large contact area in the case of protein-protein interactions, or displace the high affinity ligand) is extremely difficult. Allosteric modulators for such “undruggable” targets offer an attractive therapeutic solution. By definition, allosteric molecules bind to a site other than a protein's active site, thereby changing the protein's conformation with a concomitant functional effect (e.g. activation of a receptor). Allosteric modulation of target proteins has the added benefit of not having to rely on inhibition or competition with the binding of the natural ligand to the protein, which can result in unintended clinical side effects. However, it has been difficult to identify allosteric modulators using currently available conventional techniques. For example, structural information obtained from X-ray crystallography or NMR methods is often of limited value for drug discovery purposes due to low throughput, low sensitivity, the non-physiological conditions utilized, the size of the protein amenable to the technique, and many other factors. What is needed, therefore are high throughput techniques for screening collections of candidate compounds to rapidly identify agents capable of, for example, allosteric modulation of the target protein's conformation.
As described more fully below, second harmonic generation (SHG) is a nonlinear optical process which may be configured as surface-selective detection technique that enables detection of conformational change in proteins and other biological targets (as described previously, for example, in U.S. Pat. No. 6,953,694, and U.S. patent application Ser. No. 13/838,491). In order to deploy SHG-based detection of conformational change in a high throughput format, it may be advantageous to design novel mechanisms for rapid, precise, and interchangeable positioning of substrates (comprising the biological targets to be analyzed) with respect to the optical system used to deliver excitation light, which at the same time ensure that efficient optical coupling between the excitation light and the substrate surface is maintained. One preferred format for high throughput optical interrogation of biological samples is the glass-bottomed microwell plate.
The systems and methods disclosed herein provide mechanisms for coupling the high intensity excitation light required for SHG and other nonlinear optical techniques to a substrate, e.g. the glass substrate in a glass-bottomed microwell plate, by means of total internal reflection in a manner that is compatible with the requirements for a high throughput analysis system.